Mitochondrial apoptosis-induced channel (MAC) function triggers a Bax/Bak-dependent bystander effect.

Collateral spread of apoptosis to nearby cells is referred to as the bystander effect, a process that is integral to tissue homeostasis and a challenge to anticancer therapies. In many systems, apoptosis relies on permeabilization of the mitochondrial outer membrane to factors such as cytochrome c and Smac/DIABLO. This permeabilization occurs via formation of a mitochondrial apoptosis-induced channel (MAC) and was mimicked here by single-cell microinjection of cytochrome c into Xenopus laevis embryos. Waves of apoptosis were observed in vivo from the injected to the neighboring cells. This finding indicates that a death signal generated downstream of cytochrome c release diffused to neighboring cells and ultimately killed the animals. The role of MAC in bystander effects was then assessed in mouse embryonic fibroblasts that did or did not express its main components, Bax and/or Bak. Exogenous expression of green fluorescent protein-Bax triggered permeabilization of the outer membrane and apoptosis in these cells. Time-lapse videos showed that neighboring cells also underwent apoptosis, but expression of Bax and/or Bak was essential to this effect, because no bystanders were observed in cells lacking both of these MAC components. These results may guide development of novel therapeutic strategies to selectively eliminate tumors or minimize the size of tissue injury in degenerative or traumatic cell death.

Expression of exogenous mRNA in Xenopus laevis embryos for the study of cell cycle regulation.

The microinjection of mRNA that is transcribed and capped in vitro into fertilized eggs and embryos of Xenopus laevis provides a powerful means for discovering the function of proteins during early development. Proteins may be overexpressed for a gain-of-function effect or exogenous protein function may be compromised by the microinjection of mRNA encoding "dominant-negative" proteins. This methodology is particularly suited for the investigation of the regulation of the cell cycle, checkpoints, and apoptosis in early development.

Chk1 is activated at the midblastula transition in Xenopus laevis embryos independently of DNA content and the cyclin E/Cdk2 developmental timer.

Cell cycle checkpoints that are engaged in response to damaged and unreplicated DNA may serve additional, constitutive functions. In the developing Xenopus laevis embryo, the checkpoint kinase Chk1 is transiently activated at the midblastula transition (MBT), a period of extensive cell cycle remodeling including the acquisition of cell cycle checkpoints. The timing of many cell cycle remodeling events at the MBT, such as the lengthening of cell cycles, depends upon a critical nucleocytoplasmic (N/C) ratio. However, other events, including the degradation of maternal cyclin E, do not depend upon the N/C ratio, and are regulated by an autonomous developmental timer. To better understand what regulates Chk1 activation at the MBT, embryos were treated with aphidicolin, at different developmental times and for different lengths of time, to reduce the DNA content at the MBT. Chk1 was activated at the MBT in these embryos establishing that Chk1 activation occurs independently of the N/C ratio. Cdc25A is normally phosphorylated by Chk1 at the MBT and then degraded. The degradation of Cdc25A demonstrated partial dependence on DNA content, suggesting that factors other than Chk1 regulate its degradation. When the cyclin E developmental timer was disrupted with the Cdk2 inhibitor delta34-Xic1, Chk1 was still activated at the MBT, indicating that activation of Chk1 at the MBT was not directly linked to the cyclin E timer. Conversely, unreplicated or damaged DNA, delayed the degradation of cyclin E at the MBT, indicating that the cyclin E/Cdk2 timer is sensitive to engagement of cell cycle checkpoints.

Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of Xenopus laevis.

BACKGROUND: The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT) of early development. Cell divisions 2-12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk) activity. RESULTS: In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, Delta34-Xic1, was injected in embryos and also shown to induce apoptosis. CONCLUSION: Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and Delta34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in X. laevis embryos.

Cyclin A1/Cdk2 is sufficient but not required for the induction of apoptosis in early Xenopus laevis embryos.

The role of cyclin-dependent kinases in cell proliferation is well characterized, whereas their somewhat paradoxical role in catalyzing apoptosis is less understood. One Cdk complex implicated in both cell proliferation and cell death is cyclin A/Cdk2. During early embryonic development of Xenopus laevis, distinct isoforms of cyclin A are expressed at different times. From fertilization through gastrulation, cyclin A1 is the predominant isoform. Cyclin A1 primarily dimerizes with Cdk2 but not Cdk1. In contrast, cyclin A2 is expressed at a low level until gastrulation, when it becomes the major A-type cyclin and associates with both Cdk1 and Cdk2. When Xenopus embryos are treated with ionizing radiation (IR) prior to the midblastula transition (MBT), cyclin A1 protein persists beyond the MBT and forms an active complex with Cdk2. During this window of cyclin A1/Cdk2 activity, the embryo undergoes apoptosis. To test the hypothesis that cyclin A1-associated activity is a mediator of apoptosis, cyclin A1 protein level and associated kinase activity were measured in embryos treated with aphidicolin to induce apoptosis. Both cyclin A1 content and associated kinase activity were sustained after the MBT as embryos underwent apoptosis. To determine whether cyclin A1/Cdk2 was sufficient to induce apoptosis, recombinant cyclin A1/Cdk2 complex was injected into single-celled embryos, which induced apoptosis after the MBT. However, morpholinos targeting translation of cyclins A1 and A2 did not block apoptosis in embryos treated with X-rays or aphidicolin. These data indicate that cyclin A1/Cdk2 is sufficient, but not required for apoptosis during early development.

Chk2/Cds1 protein kinase blocks apoptosis during early development of Xenopus laevis.

Early Xenopus laevis embryos possess cell cycles that do not arrest at checkpoints in response to damaged DNA. At the midblastula transition (MBT), embryos with damaged DNA undergo apoptosis. After the MBT, DNA damage triggers cell cycle arrest rather than apoptosis. The transition from checkpoint-unregulated to checkpoint-regulated cycles makes Xenopus embryos compelling for studying mechanisms regulating response to genomic damage. The DNA damage checkpoint is mediated by the Chk2/Cds1 kinase. Conflicting evidence implicates Chk2 as an inhibitor or promoter of apoptosis. To better understand the developmental function of Chk2, we expressed wild-type (wt) and dominant-negative (DN) Chk2 in Xenopus embryos. Wt-Chk2 created a pre-MBT checkpoint due to degradation of Cdc25A and phosphorylation of cyclin-dependent kinases. Embryos expressing DN-Chk2 developed normally until gastrulation and then underwent apoptosis. Conversely, low doses of wt-Chk2 blocked radiation-induced apoptosis. Therefore, Chk2 operates at a switch between cell cycle arrest or apoptosis in response to genomic assaults.

Altered expression of Chk1 disrupts cell cycle remodeling at the midblastula transition in Xenopus laevis embryos.

Studies in several model systems, including Xenopus laevis oocytes and embryos, have indicated that the checkpoint kinase, Chk1, is required for early development, even in the absence of damaged or unreplicated DNA. Chk1 is transiently activated at the midblastula transition (MBT) in Xenopus, a time when the cell cycle remodels from rapid embryonic cleavage cycles to longer, more regulated somatic cell cycles. To better understand the role of Chk1 in cell cycle remodeling, mRNA encoding Chk1 was microinjected into 1-cell stage embryos, and the effects on both the MBT and on the expression of several cell cycle regulators were examined. Zygotic transcription, a hallmark of the MBT that depends upon the nucleocytoplasmic (N/C) ratio, was blocked, as was degradation of maternal cyclin E, an event of the MBT that occurs independent of the N/C ratio. Levels of mitotic cyclins were elevated throughout early development, consistent with cell cycle arrest at G2/M. In these embryos, Cdc25A level was low, whereas Cdc25C level was not affected. Furthermore, the level of Wee1 increased at 6 hrs post-fertilization (pf), the time at which the MBT normally occurs, even though these embryos did not demonstrate any known markers of the MBT. These studies suggest that in addition to targeting Cdc25A for degradation, Chk1 may also function in cell cycle remodeling at the MBT by stabilizing Wee1 until it is replaced by the somatic Wee2 protein during gastrulation.